The incidence and cost of treating respiratory tract allergic disease is increasing. Cost-efficient, more effective, or preventative therapeutics are therefore desirable1.
One such allergic disease is asthma in which the inflammatory pathology is predominantly mediated by cytokines which utilise a common intra-cellular transcription factor known as STAT6 (signal transducer and activator of transcription 6). STAT6 is critical for allergy development, mucosal/airway inflammation and asthma (STAT6-deficient animals do not get asthma, even when challenged in a way that induces asthma in normal mice).
Drugs that specifically and effectively target STAT6, which resides and operates in the intracellular environment, have proved difficult to develop. For example, anti-STAT6 peptides have been investigated13 but were found to achieve only limited and very transient (minutes) repression of STAT6 protein expression. The transient effect is considered to be due to peptide degradation by endogenous cellular proteases.
Attempts to repress STAT6 expression in vivo through antisense DNA techniques15 have proved unsuccessful. This approach suffers from a series of problems. For example only a low inhibition of STAT6 expression is obtained, even at high concentrations of antisense DNA, the effects are transient and the antisense molecule is subject to degradation and is difficult to target to the appropriate intracellular location. The high concentration of antisense DNA required to produce any useful effect often causes the antisense DNA to exhibit antigenic properties and can invoke an immune response. Furthermore, mice treated with STAT6 directed antisense DNA did not exhibit an improvement in allergic symptoms and developed splenomegaly16, i.e. a toxic side effect.
Accordingly, to date, STAT6 has proved to be a very difficult molecule to effectively inhibit or repress in a therapeutically useful manner. Despite several attempts, no successful drug or composition has been developed that targets STAT6 effectively without causing non-specific side-effects.
STAT6
STAT6 is the Signal Transducer and Activator of Transcription 6. To be functional in intact cells, STAT6 has to be phosphorylated. Sequence data for human STAT6 can be accessed from NCBI (www.ncbi.nlm.nih.gov) under accession numbers NP—003144 (NM—003153) and U16031.
RNA INTERFERENCE (RNAi)
RNAi utilises small double-stranded RNA molecules (dsRNA) to target messenger RNA (mRNA), the precursor molecule that cells use to translate the genetic code into functional proteins. During the natural process of RNAi, dsRNA is enzymatically processed into short-interfering RNA (siRNA) duplexes of 21 nucleotides in length. The antisense strand of the siRNA duplex is then incorporated into a cytoplasmic complex of proteins (RNA-induced silencing complex or RISC). The RISC complex containing the antisense siRNA strand also binds mRNA which has a sequence complementary to the antisense strand—allowing complementary base-pairing between the antisense siRNA strand and the sense mRNA molecule. The mRNA molecule is then specifically cleaved by an enzyme (RNase) associated with the RISC resulting in specific gene silencing3,4. For gene silencing (i.e. mRNA cleavage) to occur, anti-sense RNA (i.e. siRNA) has to become incorporated into the RISC. This is a natural and highly efficient process that occurs in all nucleated cells and whose origin is thought to be in mediating protection from transposable elements (e.g. viruses) and in normal regulation of gene expression. It is therefore distinct from the artificial process of introducing anti-sense DNA molecules into cells, where targeting of mRNA occurs through simple base-pairing of the naked anti-sense DNA molecule to its RNA target.
The advantages of RNAi over other gene-targeting strategies such as DNA anti-sense oligonucleotides can include its relative specificity, its enhanced efficacy, and the fact that siRNA treatment feeds into a natural RNAi pathway that is inherent to all cells.
However, the success of RNAi in gene repression or silencing is unpredictable, indeed the outcome can be highly variable and may depend on a variety of factors which include the accessibility of the genetic target (i.e. mRNA) and the efficiency of RNAi in the cell type being targeted.